Composition for preventing or treating mycoplasma synoviae infection

ABSTRACT

The present disclosure relates to a composition for preventing and treating  Mycoplasma synoviae  infection, the composition comprising Tuf, NOX, MS53-0285 or a combination thereof as active ingredients. The composition of the present disclosure is effective in alleviating symptoms caused by  Mycoplasma synoviae  infection and can be used as an effective tool in preventing or treating poultry diseases.

BACKGROUND Technical Field

The present application relates to a composition for preventing ortreating Mycoplasma spp. infection, particularly to a composition forpreventing or treating Mycoplasma synoviae.

Description of Related Art

Mycoplasma spp. is currently known as the smallest prokaryote capable ofself-replicating extracellularly. They are spherical with a diameter of0.3 to 0.9 μm, or filament-like with a length of 1 μm. As fastidiousbacteria, they are difficult to culture and their growth rate is veryslow. It is known that several types of Mycoplasma spp. are associatedwith a number of human or animal diseases.

Studies have shown that poultry can be infected with dozens of types ofMycoplasma spp., including Mycoplasma synoviae, a disease-causingmicroorganism listed in the OIE (World Organization for Animal Health)document regarding Mycoplasma diseases. Thus, Mycoplasma synoviae is animportant pathogen to be taken into consideration for the prevention ortreatment in poultry.

Mycoplasma synoviae may cause synovitis or arthritis. Mycoplasmasynoviae infection in broiler chicken will result in lower feedefficiency, higher mortality rate and poultry carcasses condemnationrate. Mycoplasma synoviae infection in breeder chickens and layerchickens will result in lower egg production rate and higher embryomortality rate. Mycoplasma synoviae infection in chickens will be morelikely to result in secondary infection caused by viral or bacterialpathogens, causing significant financial losses for poultry farmers.Manufacturers of veterinary vaccines worldwide have developed live andinactivated vaccines for the poultry industry; however, culturingMycoplasma synoviae is difficult and costly. Therefore, there remains aneed in this field for vaccines that can be used for preventing andtreating Mycoplasma synoviae infection.

SUMMARY

Accordingly, one object of the present disclosure is to provide antigenssuitable for being used in Mycoplasma synoviae subunit vaccines, therebyproducing subunit vaccines to reduce disease prevention costs.

Another object of the present disclosure is to provide a subunit vaccinewith multiple antigens that can be used for preventing and treatingMycoplasma synoviae infection. Such vaccine provides better protectionthan single-antigen vaccines.

To this end, the present disclosure provides a composition forpreventing Mycoplasma synoviae infection, comprising: activeingredients, which comprise Tuf, NOX, MS53-0285 and a combinationthereof, wherein said Tuf has a sequence of SEQ ID NO: 01, said NOX hasa sequence of SEQ ID NO: 02, and said MS53-0285 has a sequence of SEQ IDNO: 03; and a pharmaceutically acceptable adjuvant.

Preferably, said active ingredients are at least two proteins selectedfrom the group consisting of: Tuf, NOX and MS53-0285. More preferably,said active ingredients include Tuf, NOX and MS53-0285.

More preferably, said active ingredients have a concentration of 40 to900 μg/mL based on the total volume of said composition.

Possibly, said pharmaceutically acceptable adjuvant is a complete orincomplete Freund's adjuvant, alumina gel, surfactant, polyanionadjuvant, peptide, oil emulsion, or a combination thereof.

Preferably, said composition further comprises a pharmaceuticallyacceptable additive. Possibly, said pharmaceutically acceptable additiveis a solvent, stabilizer, diluent, preservative, antibacterial agent,antifungal agent, isotonic agent, absorption delaying agent, or acombination thereof.

Preferably, said symptoms caused by Mycoplasma synoviae infection aretracheal lesion, air sac lesion, arthritis or a combination thereof.

Preferably, said symptoms caused by Mycoplasma synoviae infection are atleast arthritis provided that said active ingredients include Tuf.Preferably, said symptoms caused by Mycoplasma synoviae infection are atleast air sac lesion provided that said active ingredients include NOXand MS53-0285, or a combination thereof. More preferably, said symptomscaused by Mycoplasma synoviae infection are at least tracheal lesionprovided that said active ingredients include MS53-0285.

Preferably, said symptoms caused by Mycoplasma synoviae infection aretracheal lesion, air sac lesion and arthritis provided that said activeingredients comprise Tuf, NOX and MS53-0285.

The present disclosure also provides an expression vector, comprising: anucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or acombination thereof; expression elements including a promoter and aribosome binding site; and a fusion partner sequence.

The present disclosure further provides an expression vector,comprising: a nucleotide sequence translated as a protein of Tuf, NOX,MS53-0285 or a combination thereof, wherein said Tuf has a sequence ofSEQ ID NO: 01, said NOX has a sequence of SEQ ID NO: 02, and saidMS53-0285 has a sequence of SEQ ID NO: 03; expression elements includinga promoter and a ribosome binding site; and fusion partner sequences.

Preferably, said fusion partner is DsbC from E. coli, MsyB from E. coli,FklB from E. coli or a combination thereof.

Preferably, said expression vector has a sequence of SEQ ID NO: 07, SEQID NO: 08, or SEQ ID NO: 09.

The present disclosure further provides the use of protein for producinga composition for preventing and treating Mycoplasma synoviae infection,wherein said protein comprises Tuf, NOX, MS53-0285 or a combinationthereof; wherein said Tuf has a sequence of SEQ ID NO: 01, said NOX hasa sequence of SEQ ID NO: 02, and said MS53-0285 has a sequence of SEQ IDNO: 03.

In view of the foregoing, the present disclosure discloses antigens usedfor preventing and treating Mycoplasma synoviae infection and as activeingredients for subunit vaccines. The results of the present disclosurenot only provide a novel option for preventing Mycoplasma synoviaeinfection, but also discloses that “cocktail” subunit vaccines combiningtwo or more antigens (i.e. containing two or more antigens as activeingredients) have a better immunity-inducing effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of Example 4 of the present invention. (A)Protein electrophoresis was used to evaluate the solubility of therecombinant antigens manufactured in the present disclosure (Trepresents total cell lysates; S represents soluble fraction). (B)Protein electrophoresis was used to evaluate the purity of therecombinant antigens purified in the present invention.

FIG. 2 shows the positive Mycoplasma synoviae-specific antibodiesresponses in the chicken serum of Example 5 of the present invention.

FIG. 3 shows the level of increase in footpad thickness of chickensinfected with Mycoplasma synoviae in Example 5 of the present invention.

FIG. 4 shows the level of increase in footpad temperature of chickensinfected with Mycoplasma synoviae in Example 5 of the present invention.

FIG. 5 shows the footpad infection scores of chickens infected withMycoplasma synoviae in Example 5 of the present invention.

FIG. 6 shows (A) the air sac lesion scores of chickens infected withMycoplasma synoviae in Example 5 of the present invention, and (B)tracheal mucosal thickness of chickens in Example 5. The statisticsshows that the vaccines of the present disclosure are statisticallysignificant compared to the positive control group (T-test, †: p<0.1).

FIG. 7 shows the positive Mycoplasma synoviae-specific antibodiesresponses in the chicken serum of Example 6 of the present invention.

FIG. 8 shows the positive Mycoplasma synoviae-specific antibodiesresponses in the chicken serum of Example 7 of the present invention.

FIG. 9 shows the average daily gain of chickens infected with Mycoplasmasynoviae in Example 7 of the present invention.

FIG. 10 shows footpad thickness changes displayed by chickens infectedwith Mycoplasma synoviae in Example 7 of the present invention. Thestatistics shows that the vaccines of the present disclosure arestatistically significant compared to the positive control group(T-test, †: p<0.1).

FIG. 11 shows footpad temperature changes displayed by chickens infectedwith Mycoplasma synoviae in Example 7 of the present invention. Thestatistics shows that the vaccines of the present disclosure arestatistically significant compared to the positive control group(T-test, †: *: p<0.05).

FIG. 12 shows the positive Mycoplasma synoviae-specific antibodiesresponses in the chicken serum of Example 8 of the present invention.

FIG. 13 shows the average daily gain of chickens infected withMycoplasma synoviae in Example 8 of the present invention. Thestatistics shows that the vaccines of the present disclosure arestatistically significant compared to the positive control group(T-test, †: *: p<0.05).

FIG. 14 shows footpad temperature changes displayed by chickens infectedwith Mycoplasma synoviae in Example 8 of the present invention. Thestatistics shows that the vaccines of the present disclosure arestatistically significant compared to the positive control group(T-test, †: p<0.1; *: p<0.05).

DETAILED DESCRIPTION

The development results of the present disclosure prove that Tuf, NOXand MS53-0285 can be used as active ingredients in subunit vaccines forpreventing and treating Mycoplasma synoviae infection. The phrase“preventing and treating Mycoplasma synoviae infection” herein refers toreduce infection level of Mycoplasma synoviae in poultry, such asalleviating diseases caused by Mycoplasma synoviae infection. Saiddiseases include but not limited to tracheal lesion, air sac lesion orarthritis (such as footpad swelling or footpad inflammation). From ascientific point of view, it is impossible to demonstrate whether thesubjects are not infected with said disease-causing microorganisms atall. Therefore, it can be understood by a person having ordinary skillin the art that in the field of disease prevention, the act of“preventing and treating” does not intend to keep the subject from beinginfected with any of said disease-causing microorganisms.

In one aspect, the present disclosure relates to a composition forpreventing and treating Mycoplasma synoviae infection. In one preferredembodiment, the composition is a subunit vaccine. In one preferredembodiment, the composition comprises Tuf as active ingredients, andsaid Tuf has a sequence of SEQ ID NO: 01. In another preferredembodiment, the composition comprises NOX as active ingredients, andsaid NOX has a sequence of SEQ ID NO: 02. In yet another preferredembodiment, the composition comprises MS53-0285 as an active ingredient,and said MS53-0285 has a sequence of SEQ ID NO: 03.

A person having ordinary skill in the art can readily understand that aslong as the antigen determinants on said proteins are not affected, saidamino acid sequence can be altered to a certain degree and still fallswithin the scope of the present invention. For example, a few aminoacids in said sequence may be altered by a person having ordinary skillin the art, but the immunity-inducing effect as described herein canstill be generated. Alternatively, based on the needs of a person havingordinary skill in the art, other sequences may be added into saidsequence to facilitate production without affecting theimmunity-inducing effect as described herein. In this regard, themodified sequence should still falls within the scope of the presentinvention.

In one embodiment, when said active ingredients include Tuf, saidsymptoms caused by Mycoplasma synoviae infection are at least arthritis.Nevertheless, the fact that said active ingredients include Tuf does notsuggest that they are ineffective in preventing other diseases, butrather that they are more effective in preventing arthritis.

In one embodiment, when said active ingredients include NOX, MS53-0285or a combination thereof, said symptoms caused by Mycoplasma synoviaeinfection are at least air sac lesion. Nevertheless, the fact that saidactive ingredients include NOX, MS53-0285 or a combination thereof doesnot suggest that they are ineffective in preventing other diseases, butrather that they are more effective in preventing air sac lesion.

In one embodiment, when said active ingredients include MS53-0285, saidsymptoms caused by Mycoplasma synoviae infection are at least tracheallesion. Nevertheless, the fact that said active ingredients includeMS53-0285 does not suggest that they are ineffective in preventing otherdiseases, but rather that they are more effective in preventing tracheallesion.

In one embodiment, when said active ingredients include Tuf, NOX andMS53-0285, said symptoms caused by Mycoplasma synoviae infection are atleast tracheal lesion, air sac lesion and arthritis.

In another aspect, the present disclosure relates to a cocktail vaccineused for preventing or treating Mycoplasma synoviae infection. Thecocktail vaccine as described herein contains two and more antigens. Ina preferred embodiment, the cocktail vaccine as described hereincontains two and more antigens from the same type of disease-causingmicroorganism. In general, combining two or more antigens in a singlevaccine does not necessarily contribute synergistically to theimmunity-inducing effect of the vaccine. Instead, it may lead to anunfavorable consequence that the immunity-inducing effects of two ormore antigens offset each other. In terms of cost, even if theimmunity-inducing effects of two or more antigens do no offset eachother, it is not worthwhile combining these two or more antigens in asingle vaccine when there is no synergistic effect.

In a preferred embodiment, the active ingredients of said cocktail areat least two proteins selected from the group consisting of thefollowing proteins: Tuf, NOX and MS53-0285. More preferably, said activeingredients include Tuf, NOX and MS53-0285. In a possible embodiment, aslong as the antigen determinants on the folding from said amino acidsequences are not destroyed, said active ingredients may be fusionproteins having any two or more of said amino acid sequences. In anotherpossible embodiment, said cocktail vaccine uses said proteins that areindependent of each other.

In a preferred embodiment, the composition of the present disclosurefurther comprises a pharmaceutically acceptable adjuvant and/or apharmaceutically acceptable additive. Said pharmaceutically acceptableadjuvant is used for enhancing the immunity-inducing effect of activeingredients, maintaining the stability of active ingredients, andenhancing the safety of said composition when used as a vaccine. Saidpharmaceutically acceptable adjuvant is, including without limitation, acomplete or incomplete Freund's adjuvant, alumina gel, surfactant,polyanion adjuvant, peptide, oil emulsion, or a combination thereof.Said pharmaceutically acceptable additive may be, including withoutlimitation, a solvent, stabilizer, diluent, preservative, antibacterialagent, antifungal agent, isotonic agent, absorption delaying agent, or acombination thereof.

In a possible embodiment, the composition of the present disclosure canbe made into formulations that are administered orally, nasally,intravenously, intramuscularly or intraperitoneally. In a possibleembodiment, the active ingredients in the composition of the presentdisclosure have a concentration of 40 to 900 μg/mL based on the totalvolume of said composition. In another possible embodiment, the activeingredients in the composition of the present disclosure have aconcentration of 40 to 600 μg/mL based on the total volume of saidcomposition. In the cocktail vaccine of the present invention, theconcentration of said active ingredients is the total concentration ofall the antigens contained in said cocktail vaccine. In one embodiment,for example, when the cocktail vaccine of the present disclosurecontains said Tuf antigens and said NOX antigens as active ingredients,said “concentration of active ingredients” is the total of theconcentration of said Tuf antigens and that of said NOX antigens. Inanother embodiment, when the cocktail vaccine of the present disclosurecontains said Tuf antigens, said NOX antigens and said MS53-0285 asactive ingredients, said “concentration of active ingredients” is thetotal of the concentration of said Tuf antigens, that of said NOXantigens, and that of MS53-0285 antigens.

On the other hand, an obstacle to express said active ingredients inprokaryotic expression systems is to purify the expressed activeingredients from the said prokaryotic expression systems. Recombinantproteins expressed in prokaryotic expression systems are typicallynon-soluble, thereby making the purification process more difficult andcostly. In this regard, one advantage of the antigen expression vectorof the present disclosure is the ability to express soluble recombinantantigens, thereby simplifying the purification process and reducing itscosts.

Thus, in another aspect, the present disclosure relates to an expressionvector, which is used for producing said Tuf, NOX or MS53-0285. Theexpression vectors of the present disclosure comprise nucleotidesequences, expression elements and fusion partner sequences. Saidnucleotide sequences can be translated as Tuf, NOX, MS53-0285 or acombination thereof.

Said expression elements refer to elements required for initiatingtranscription and translation processes in the expression system. Saidexpression elements should at least include a promoter and a ribosomebinding site. Preferably, said expression elements may further include:an operator, enhancer sequences for enhancing translation/transcription,or a combination thereof. In a preferred embodiment, said expressionvector is used in an E. coli expression system, and said expressionelements can be recognized by E. coli.

In a preferred embodiment, the research of the present disclosure hasproved that DsbC from E. coli, MsyB from E. coli, FklB from E. coli, ora combination thereof is preferably used as a fusion partner forexpression of said antigens of the present invention, thereby making therecombinant proteins produced in a prokaryotic expression system assoluble form. In a preferred embodiment, in order to facilitate thepurification process, said expression vector may further comprise asequence encoding His-tag, GST-tag or a combination thereof, so that theobtained recombinant proteins can be purified using a nickel ionaffinity column or a glutathione affinity column.

In yet another aspect, the present disclosure relates to a use ofcomposition, in which said Tuf, NOX or MS53-0285 are used, forpreventing and treating Mycoplasma synoviae infection. No furtherelaboration is needed for said composition for preventing and treatingMycoplasma synoviae infection since it has been previously described.

The following examples describe the experiments carried out for thedevelopment of the present disclosure to further illustrate the featuresand advantages thereof. It should be understood that the examples listedbelow are merely exemplary of the invention, and should not be used tolimit the scope of the claims.

Example 1: Cloning of Mycoplasma synoviae Genes

Gene-specific primers are designed for various antigens (Table 1). Agenomic DNA of Mycoplasma synoviae was used as a template andgene-specific primers were combined to amplify target genes.

TABLE 1 Primer pairs used for amplifying target genes TargetPrimer sequence gene Primer sequence (5′ to 3′) tufTUFBAMHIF (SEQ ID NO: 10): GATATAGGATCCATGGCAAAATTAGATTTTGACCGTTTUFSALIR (SEQ ID NO: 11): CAATATGTCGACTTTAACGATTTTTGTAACTGATCCGG noxNOXBAMHIF (SEQ ID NO: 12): GATATAGGATCCATGGAAAACAAAAAAATTATAGTTGT TGGTNOXSALIR (SEQ ID NO: 13): CAATATGTCGACAGCTTTATATTTTAAACCAAGTGCTC TTAAAms53- MS53BAMHIF (SEQ ID NO: 14): 0285GATATAGGATCCATGAATAAAACAAAAATTAAATTTAT TTTAGGAAMS53SALIR (SEQ ID NO: 15): CAATATGTCGACATTATTATTTGAACCAAATGTATCTCTAAATGA *GGATCC: BamHI cleavage site; GTCGAC: SalI cleavage site

1. Extracting Genomic DNA of Mycoplasma synoviae

-   -   The genomic DNA of Mycoplasma synoviae WVU 1853 (American Type        Culture Collection® 25204™) was extracted using a DNA        purification kit (Tissue & Cell Genomic DNA Purification kit;        GMbiolab, Taiwan). First, 4.5 mL of the liquid culture of        Mycoplasma synoviae was added into a centrifuge tube. After        centrifugation (5,870×g, 5 minutes), the supernatant was removed        and the pellet was collected. Then, 20 μL of proteinase K (10        mg/mL) and 200 μL of extraction reagent were added and allowed        to react at 56° C. for 3 hours. 200 μL of binding solution was        added and allowed to react at 70° C. for 10 minutes. When the        reaction was completed, 200 μL of absolute alcohol was added and        the mixture was transferred into a micro-centrifuge tube to be        mixed thoroughly. The resulting solution (including        precipitates) was pipetted into a spin column and the spin        column was then put into a collection tube. After centrifugation        (17,970×g) for 2 minutes, the supernatant was removed and 300 μL        of binding solution was added again into the spin column. After        centrifugation (17,970×g) for 2 minutes, the supernatant was        removed. 700 μL of wash solution was added into the spin column        and the mixture was centrifuged (17,970×g) for 2 minutes, and        then the supernatant was removed, with the above steps being        repeated once. Finally, the mixture was centrifuged at 17,970×g        for 5 minutes to remove residual alcohol. The spin column was        put into a sterilized micro-centrifuge tube and an appropriate        amount of sterile deionized water was added to drain the genomic        DNA.

2. Amplifying Target Genes Using Polymerase Chain Reaction (PCR)

-   -   The genomic DNA of Mycoplasma synoviae was used as a template        and primers designed for tuf gene, nox gene, and ms53-0285 gene        were used for PCR reaction, respectively. Primers used in PCR        and PCR reaction conditions are shown in Table 2 below (primer        sequences are shown in Table 1), wherein 50 μL of PCR reaction        mixture contained 1×GDP-HiFi PCR buffer B, 200 μM of mixture of        dATP, dTTP, dGTP, and dCTP, 1 μM of amplification primers, 200        ng of Mycoplasma synoviae genomic DNA and 1 U of GDP-HiFi DNA        polymerase. After the completion of the PCR reaction, gel        electrophoresis was conducted to confirm the existence of the        DNA fragments with expected sizes.

TABLE 2 PCR reaction conditions and primer pairs Target gene Primer pairReaction conditions tuf TUFBAMHIF 96° C. for 5 minutes; 94° C. for 30TUFSALIR seconds, 55° C. for 30 seconds, 68° C. for 45 seconds (35cycles); 68° C. for 5 minutes nox NOXBAMHIF 96° C. for 5 minutes; 94° C.for 30 NOXSALIR seconds, 55° C. for 30 seconds, 68° C. for 45 seconds(35 cycles); 68° C. for 5 minutes ms53-0285 MS53BAMHIF 96° C. for 5minutes; 94° C. for 30 MS53SALIR seconds, 55° C. for 30 seconds, 68 C.for 1 minute and 15 seconds (35 cycles); 68° C. for 5 minutes

3. Recovery and Cloning PCR Products

-   -   The recovery of the PCR products was carried out using the PCR        Clean Up system kit (GMbiolab, Taiwan) and following the        operations manual. Cloning of the target gene was then carried        out using the CloneJET PCR Cloning Kit according to the        manufacturer's operations manual. The ligation mixture was        transformed into E. coli strain ECOS™ 9-5. The conditions of        transformation are provided in the manual or may be modified        based on standard experimental procedures in the art.    -   The bacteria transformed were cultured on LB solid medium        containing ampicillin (100 μg/mL). After the formation of        colonies, a colony PCR was conducted to screen the strains.        First, lx Taq reaction buffer, 200 μM dNTP (dATP, dTTP, dGTP and        dCTP), 1 μM amplification primers and 2.5 U DreamTaq DNA        polymerase (Thermo, USA) were mixed to formulate 100 μL PCR        reagent. After mixing, the PCR mixture was distributed to        several PCR tubes (10 μL/tube). Colonies on the plates were        picked into the PCR tubes for PCR reaction. The conditions for        PCR reaction are: 95° C. for 5 minutes; 95° C. for 30 seconds,        55° C. for 30 seconds, 72° C. for X minutes (25 cycles); 72° C.        for 7 minutes, wherein “X” was determined by the extension time        required for the DNA polymerase. The time for tuf gene and nox        gene was set as 1 minute 30 seconds, and the time for ms53-0285        gene was set as 2 minutes 30 seconds. After the PCR reaction,        gel electrophoresis was conducted to confirm the PCR results.        Colony PCR was used to confirm that recombinant plasmids in the        transformed strains had insert DNA, and plasmids therein were        then extracted for DNA sequencing (Tri-I Biotech, Inc.). The        plasmids having tuf gene, nox gene and ms53-0285 gene were named        as pJET-TUF, pJET-NOX and pJET-MS53, respectively.

4. Site-Directed Mutagenesis of Nox Gene

-   -   nox gene has three TGA codons. TGA codons can be translated as        tryptophan in Mycoplasma spp., and as stop codons in E. coli. In        order to avoid failure to produce intact proteins using E. coli        expression system, point mutations at TGA codons in nox gene        must be induced, in order to replace said codons with those        (TGG) that can be recognized by E. coli and translated as        tryptophan.    -   Criteria for designing mutagenic primers included centering the        mutation in the middle of the primer with a Tm of at least        78° C. The Tm value of the primer was calculated using the        following formula provided by Invitrogene:

Tm=81.5+0.41(% GC)−675/N−% mismatch,

where “% GC” is the percentage of G or C nucleotides in the primer; “N”is the length of the primer; and “% mismatch” is the percentage ofmutated bases in the primer. The primers used for point mutation of noxgene are listed in Table 3 below, including NOXBAMHIF/NOXM2,NOXM1/NOXM4, NOXM3/NOXM6 and NOXM5/NOXSALIR.

TABLE 3 Primers used for point mutation in nox gene PrimerPrimer sequence (5′ to 3′) NOXBAMHIF SEQ ID NO: 16GATATAGGATCCATGGAAAACAAAAAAATTATAGTTGTTGGT NOXM2 SEQ ID NO: 17

NOXM1 SEQ ID NO: 18

NOXM4 SEQ ID NO: 19

NOXM3 SEQ ID NO: 20

NOXM6 SEQ ID NO: 21

NOXM5 SEQ ID NO: 22

NOXSALIR SEQ ID NO: 23 CAATATGTCGACAGCTTTATATTTTAAACCAAGTGCTCTTAAA

-   -   The 50 μL of PCR mixture contained 1×GDP-HiFi PCR buffer B; 200        μM of mixture of dATP, dTTP, dGTP, and dCTP; 1 μM of        amplification primer; 100 ng of pJET-NOX and 1 U GDP-HiFi DNA        polymerase. The PCR reaction condition was: 98° C. for 2        minutes; 94° C. for 30 seconds, 55° C. for 30 seconds, 68° C.        for 30 seconds (35 cycles); 68° C. for 5 minutes.    -   After the PCR reaction, gel electrophoresis was conducted to        confirm the existence of the DNA fragments with expected sizes.        The PCR products were collected using Gel-M™ gel extraction        system kit. The four PCR products collected were used as        templates for gene amplification using NOXBAMHIF/NOXSALIR primer        pair. The PCR reaction condition was: 96° C. for 2 minutes;        94° C. for 30 seconds, 55° C. for 30 seconds, 68° C. for 45        seconds (35 cycles); 68° C. for 5 minutes. Following this step,        a full-length point-mutated nox gene was obtained. PCR product        was collected using PCR Clean Up kit.

5. Site-Directed Mutagenesis of Ms53-0285 Gene

-   -   ms53-0285 gene has six TGA codons. The primers used for point        mutation of ms53-0285 gene are listed in Table 4 below,        including MS53BAMHIF/MS53M2, MS53M1/MS53M4, MS53M3/MS53M6,        MS53M5/MS53M8, MS53M7/MS53M10, MS53M9/MS53M12 and        MS53M11/MS53SALIR.

TABLE 4 Primers used for point mutation in ms53-0285 gene PrimerPrimer sequence (5′ to 3′) MS53BAMHIF SEQ ID NO:  24GATATAGGATCCATGAATAAAACAAAAATTAAATTTATTTTAGGAA MS53M2 SEQ ID NO: 25:

MS53M1 SEQ ID NO: 26

MS53M4 SEQ ID NO: 27

MS53M3 SEQ ID NO: 28

MS53M6 SEQ ID NO: 29

MS53M5 SEQ ID NO: 30

MS53M8 SEQ ID NO: 31

MS53M7 SEQ ID NO: 32

MS53M10 SEQ ID NO: 33

MS53M9 SEQ ID NO: 34

MS53M12 SEQ ID NO: 35

MS53M11 SEQ ID NO: 36

MS53SALIR SEQ ID NO: 37 CAATATGTCGACATTATTATTTGAACCAAATGTATCTCTAAATGA

-   -   The 50 μL of PCR mixture contained 1×GDP-HiFi PCR buffer B; 200        μM of mixture of dATP, dTTP, dGTP, and dCTP; 1 μM of        amplification primer; 100 ng of pJET-MS53 and 1 U GDP-HiFi DNA        polymerase. The PCR reaction condition was: 96° C. for 2        minutes; 94° C. for 30 seconds, 55° C. for 30 seconds, 68° C.        for 30 seconds (35 cycles); 68° C. for 5 minutes.    -   After the PCR reaction, gel electrophoresis was conducted to        confirm the existence of the DNA fragments with expected sizes.        The PCR products were collected using Gel-M™ gel extraction        system kit. The seven PCR products collected were used as        templates for gene amplification using MS53BAMHIF/MS53SALIR        primer pair. The PCR reaction condition was: 96° C. for 2        minutes; 94° C. for 30 seconds, 55° C. for 30 seconds, 68° C.        for 1 minute and 15 seconds (35 cycles); 68° C. for 5 minutes.        Following this step, a full-length point-mutated ms53-0285 gene        was obtained. PCR product was collected using PCR Clean Up kit.

Following the above operations, tuf gene (SEQ ID NO: 04), nox gene (SEQID NO: 05) and ms53-0285 gene (SEQ ID NO: 06) were obtained. They can betranslated as Tuf (SEQ ID NO: 01), NOX (SEQ ID NO: 02) and MS53-0285(SEQ ID NO: 03), respectively.

Example 3: Constructing Mycoplasma synoviae Antigen Expression Plasmids

Plasmids having dsbC gene of E. coli were used as backbone forconstructing Mycoplasma synoviae antigen expression plasmids. Theconstruction processes of expression plasmids are as follows:

1. Construction of Tuf Expression Vector

-   -   Amplified tuf gene was cut with BamHI and SalI and the resulting        DNA fragment was joined by T4 DNA ligase with a DsbC fusion        expression plasmid pET-HISDsbC pre-cut by the same restriction        enzymes. The ligation product was then transformed into E. coli        ECOS 9-5. Transformants were screened by colony PCR. Gel        electrophoresis was conducted to confirm the existence of the        amplified DNA fragment with the expected size. When it was        confirmed that the transformants contained inserted DNA in their        recombinant plasmids, the plasmids were extracted out for DNA        sequencing. The plasmids having the correct DNA sequence were        named as pET-HISDsbC-MSTUF (SEQ ID NO: 07).

2. Construction of NOX Expression Plasmid

-   -   Mutated nox gene was cut with BamHI and SalI and the resulting        DNA fragment was joined by T4 DNA ligase with a DsbC fusion        expression plasmid pET-DsbC pre-cut by the same restriction        enzymes. The ligation product was then transformed into E. coli        ECOS 9-5. Transformants were screened by colony PCR. Gel        electrophoresis was conducted to confirm the existence of the        amplified DNA fragment with the expected size. When it was        confirmed that the transformants contained inserted DNA in their        recombinant plasmids, the plasmids were extracted out for DNA        sequencing. The plasmids having the correct DNA sequence were        named as pET-DsbC-MSNOX (SEQ ID NO: 08).

3. Construction of MS53-0285 Expression Plasmid

-   -   Mutated ms53-0285 gene was cut with BamHI and SalI and the        resulting DNA fragment was joined by T4 DNA ligase with a DsbC        fusion expression plasmid pET-DsbC pre-cut by the same        restriction enzymes. The ligation product was then transformed        into E. coli ECOS 9-5. Transformants were screened by colony        PCR. Gel electrophoresis was conducted to confirm the existence        of the amplified DNA fragment with the expected size. When it        was confirmed that the transformants contained inserted DNA in        their recombinant plasmids, the plasmids were extracted out for        DNA sequencing. The plasmids having the correct DNA sequence        were named as pET-DsbC-MS53 (SEQ ID NO: 09).

Example 4: Expressing and Purifying Recombinant Mycoplasma synoviaeAntigens

The plasmids for expressing Mycoplasma synoviae antigens weretransformed into E. coli BL21 (DE3), respectively. A single colony ofthe strain expressing the antigens was selected and inoculated in 12 mLof LB medium containing kanamycin (final concentration: 30 μg/mL). Theresulting mixture was cultured overnight at 37° C. and at 180 rpm. Then,10 mL of the liquid culture was added to 1 L of LB medium containingkanamycin (final concentration: 30 μg/mL) and was shake-cultured (37°C., 180 rpm) until OD₆₀₀ reached about 0.4-0.6. 0.1 mM IPTG was thenadded at 28° C. to induce protein expression. After for 4 hours ofinduction, the culture was centrifuged (10,000×g, 10 minutes, 4° C.) andbacterial cell pellet was collected. The bacterial cell pellet wasresuspended in 10 mL of phosphate buffer (20 mM sodium phosphate, 500 mMNaCl, pH 7.4) and was disrupted by ultrasonication. The suspension wascentrifuged (30,966×g, 30 min) and the supernatant was collected.Finally, the supernatant was filtered through a filter membrane with0.22 μM pore size. Protein electrophoresis was conducted to observe theexpression status and solubility of the recombinant antigens.

Protein purification was then conducted using immobilized-metal ionaffinity chromatography by way of the covalent bonding between His tagon the recombinant antigens and nickel ions or cobalt ions. Therecombinant antigens were purified by ÄKTA prime plus (GE Healthcare,Sweden) equipped with 5 mL HiTrap™ Ni excel column (GE Healthcare,Sweden). First, said supernatant was introduced into HiTrap™ Ni excelcolumn after the column was equilibrated with 25 mL of phosphate buffersolution. After the sample was fully introduced, 100 mL of a wash buffersolution containing 30 mM imidazole (20 mM sodium phosphate, 500 mMNaCl, 30 mM imidazole, pH 7.4) was used to wash the column to removenon-specific proteins adhered thereon. Finally, 150 mL of an elutionbuffer solution containing 250 mM imidazole (20 mM sodium phosphate, 500mM NaCl, 250 mM imidazole, pH7.4) was used to wash off the recombinantantigens from the resin, wherein imidazole of high concentration cancompete with the recombinant antigens for binding sites on the resin,thereby causing the recombinant antigens to be washed off. The purifiedantigen solution was put into Amico™ ultra-15 ultracel-30K centrifugetube (Merck Millipore, USA) to be centrifuged (2,600×g) at 4° C. untilit reached an appropriate volume, and was then stored at 4° C. forfurther use.

The result is shown in FIG. 1. It can be observed from FIG. 1A that theexpression antigens of this example have excellent solubility so as tobe subsequently purified and commercialized. FIG. 1B shows that theantigens obtained through purification have reliable purity.

Example 5: Single-Antigen Vaccine Production and Chicken Experiments

4-week-old specific pathogen-free chickens (purchased from the AnimalDrugs Inspection Branch of the Animal Health Research Institute, Councilof Agriculture, Executive Yuan, Taiwan) were used and kept in animalhouse facilities at the inspection branch. Recombinant Mycoplasmasynoviae antigens Tuf, NOX and MS53-0285 were mixed individually with acommercially available adjuvant MONTANIDE™ Gel 01 (Seppic) following theoperations manual, so that the final dose of each single-antigen vaccinebecame 50 μg/0.5 mL. 0.5 mL of these single-antigen vaccines wereadministered subcutaneously in the back of the neck of 4-week-oldchickens. Second vaccination was conducted to chickens reaching 6 weeksof age. After vaccination, the chickens were observed for two weeks.Blood samples were then collected and centrifuged (2,000×g, 4° C., 15minutes) to obtain serum samples. Mycoplasma synoviae antibodies wereassayed using a commercial kit (IDEXX Mycoplasma synoviae Antibody TestKit).

The results are shown in Table 5 below and in FIG. 2. The specificantibodies in serum of chickens administered with single-antigenvaccines of Tuf, NOX and MS53_0285 according to the present inventionwere all positive, with a positive rate of 57%, 71% and 75%,respectively.

TABLE 5 Positive rates of single-antigen vaccines Group Control Scoregroup Tuf NOX MS53-0285 S/P ratio < 0.5 7 3 2 2 S/P ratio > 0.5 0 4 5 6S/P positive rate (%) 0 57 71 75

Next, 8-week-old chickens were challenged with Mycoplasma synoviaethrough tracheae and footpads. The challenge strain used was ATRI 2014(hereinafter “MS-f1”), which was isolated from commercial native chickenfarm in Taiwan. The challenge strain was cultured in Mycoplasma mediumat 32° C. and at 75 rpm for 32 hours.

In trachea challenge, the chickens were held and a feeding tube wasinserted into the trachea of the chickens. 1 mL of broth of challengestrain with 10⁶ CCU was then injected using a syringe. Finally, thenecks of the chickens were rubbed gently so that the broth could bespread out evenly. In footpad challenge, footpads were disinfected withalcohol wipes and 0.5 mL of broth of challenge strain with 10³ CCU wasinjected therein using a needle. After challenge, blood samples werecollected and serum was isolated to assess Mycoplasma synoviae-specificantibodies. Weight, footpad temperature and footpad thickness of thechickens at various time points were also recorded. At the end of theexperiment, a dissection was performed to observe the symptoms oftracheae, air sac and footpad swelling among the chickens. Observedresults were recorded following the diagnostic criteria for organdysfunction as described in Table 6 below.

TABLE 6 Diagnostic criteria for organ dysfunction in poultry Tracheallesion index Score Symptoms 0 No visible change. 1 Increased mucus;moderate mucosal blood stasis. 2 Increased mucus; mucosal blood stasis;cottage cheese-like discharge. 3 Increased mucus; mucosal blood stasis;cottage cheese-like discharge. Air sac lesion index Score Symptoms 0 Novisible change. 1 Slightly cloudy or covered with small yellowcheese-like fiber protein spots. 2 Cloudy; slight thickened walls;covered with yellow substance. 3 Thicker walls; cloudy and opaque;covered with discharge forming yellow spots. Arthritis (footpadswelling) Score Symptoms 0 No change compared to left footpad. 1 Mildswelling. 2 Increased swelling that causes creases on footpad surface todisappear. 3 Increased swelling that spreads to neighboring tissues. 4More severe swelling; redness and cracked skin; watery discharge/pusoozing from swollen sites that are cut open.

It could be observed from the footpad thickness of the chickens that thegroup of chickens administered with single-antigen vaccine had milderswelling than the positive group (see FIG. 3). On the other hand, thefootpad temperature measurement results showed that the single-antigenvaccine of the present disclosure alleviated chicken footpadinflammation (FIG. 4). Further assessments and records of footpadinfection showed that the chickens administered with the single-antigenvaccine of the present disclosure had less footpad infection levels,with Tuf vaccine being the most effective (FIG. 5).

Air sac lesion is another common type of infection as a result ofMycoplasma synoviae infection. Air sacs in healthy chicken should betransparent without being covered with discharges. FIG. 6A shows thatamong the chickens that had not been administered with saidsingle-antigen vaccines, their air sacs thickened and were covered withyellow cheese-like fiber protein in a visible manner. On the other hand,air sac lesion could be effectively prevented by administering thesingle-antigen vaccine of the present invention. The results also showedthat NOX and MS53-0285 vaccines were particularly effective inpreventing air sac lesion.

Chicken tracheae were further sectioned. H&E staining was performed toobserve the changes of mucus thickness on tracheal rings. The resultsshowed that mucosal infection could be effectively prevented among thevaccinated groups, compared to the non-vaccinated challenge chickens, byadministering the single-antigen vaccine of the present invention, withMS53-0285 subunit vaccine being the most effective (FIG. 6B).

It can be understood from the above results that despite slightdifferences in efficacy, Tuf, NOX and MS53-0285 vaccines of the presentdisclosure are generally effective in alleviating symptoms induced byMycoplasma synoviae.

Example 6: Multi-Antigen Vaccine (Cocktail Vaccine) Production andChicken Experiments

4-week-old specific pathogen-free chickens (purchased from the AnimalDrugs Inspection Branch of the Animal Health Research Institute, Councilof Agriculture, Executive Yuan, Taiwan) were used and kept in animalhouse facilities at the inspection branch. Various combinations ofantigens (Tuf+NOX, Tuf+MS53-0285, NOX+MS53-0285 and Tuf+NOX+MS53-0285)were mixed individually with commercially available adjuvants to obtaincocktail vaccines. Each antigen in each dose of vaccine (0.5 mL) was 50μg.

0.5 mL of said recombinant-antigen vaccine was administeredsubcutaneously in the back of the neck of 4-week-old chickens. Secondvaccination was conducted to chickens reaching 6 weeks of age. Aftervaccination, the chickens were observed for two weeks. Blood sampleswere then collected and centrifuged (2,000×g, 4° C., 15 minutes) toobtain serum samples. Mycoplasma synoviae-specific antibodies weredetected using a commercial kit (IDEXX Mycoplasma synoviae Antibody TestKit).

The results are shown in Table 7 below and in FIG. 7, suggesting thatdouble-antigen and triple-antigen vaccines both improved antibodyproduction levels in chickens. In other words, when used incombinations, recombinant antigens did not cause mutual interference;instead, they produced synergistic effect that could strengthenimmunity-inducing potential.

TABLE 7 Score S/P ratio < S/P ratio > S/P positive Group 0.5 0.5 rate(%) Tuf + NOX 3 4 57 Tuf + MS53-0285 0 7 100 NOX + MS53-0285 0 8 100Tuf + NOX + MS53-0285 1 7 88

Example 7: Multi-Antigen Vaccine (Cocktail Vaccine) Production andChicken

Experiments 4-week-old specific pathogen-free chickens (purchased fromthe Animal Drugs Inspection Branch of the Animal Health ResearchInstitute, Council of Agriculture, Executive Yuan, Taiwan) were used andkept in animal house facilities at the inspection branch. Therecombinant antigens Tuf, NOX and MS53-0285 were mixed with acommercially available adjuvant MONTANIDE™ ISA 71 VG (Seppic) to obtainthe cocktail vaccine of the present invention. Each antigen in each doseof vaccine (0.5 mL) was 50 μg. Said adjuvant was mixed with saidantigens at a 7:3 mix ratio by weight. Given that the specific gravityof said adjuvant is about 0.816, the vaccine produced in this experimentcontained approximately 0.37 mL of adjuvant.

0.5 mL of said recombinant-antigen vaccine was administeredsubcutaneously in the back of the neck of 4-week-old chickens. Secondvaccination was conducted to chickens reaching 6 weeks of age. Aftervaccination, the chickens were observed for two weeks. Blood sampleswere then collected and centrifuged (2,000×g, 4° C., 15 minutes) toobtain serum samples. Mycoplasma synoviae-specific antibodies weredetected using a commercial kit (IDEXX Mycoplasma synoviae Antibody TestKit).

The results are shown in Table 8 below and in FIG. 8, suggesting thattriple-antigen vaccines induced antibody production in chickens. Inother words, when used in combinations, recombinant antigens did notcause mutual interference; instead, they produced synergistic effectthat could strengthen immunity-inducing potential. These results are inline with those shown in FIG. 7.

TABLE 8 Group Control Triple-antigen Score group vaccine S/P ratio < 0.58 1 S/P ratio > 0.5 0 7 S/P positive rate (%) 0 88

Next, 8-week-old chickens were then challenged with Mycoplasma synoviaeMS-f1 through tracheae and footpads. After the challenge, blood sampleswere collected and serum was isolated to assess Mycoplasmasynoviae-specific antibodies. Weight, footpad temperature, and footpadthickness of each chicken at various time points were also recorded. Atthe end of the experiment, a dissection was performed to observe thesymptoms of tracheae, air sac and footpad swelling among the chickens.Observed results were recorded following the diagnostic criteria fororgan dysfunction as described in Table 6.

The results showed that the triple-antigen vaccine of the presentdisclosure could alleviate weight loss that occurred on infectedchickens (FIG. 9). In addition, the triple-antigen vaccine was alsoeffective in reducing symptoms regarding footpad swelling andtemperature (FIGS. 10 and 11). The chickens were further sacrificed anddissected. It was observed from footpad and air sac lesion scores thatthe triple-antigen vaccine was effective in lowering the severity offootpad and air sac lesion. See Table 9 below.

TABLE 9 Group Triple-antigen Score Naive vaccine Challenge Positive rateof footpad 0 88 100 infection (%) Positive rate of air sac 0 50 88lesion (%)

Example 8: Dosage Test for Triple-Antigen Vaccine

The recombinant antigens were mixed with a commercially availableadjuvant MONTANIDE™ ISA 71 VG (Seppic) to formulate subunit vaccineshaving recombinant antigens at four concentrations: 150 μg, 100 μg, 50μs and 20 μs per dose (0.5 mL), respectively. Said adjuvant was mixedwith said antigens at a 7:3 mix ratio by weight. Given that the specificgravity of said adjuvant is about 0.816, the vaccine produced in thisexperiment contained approximately 0.37 mL of adjuvant. Groups weredesigned as shown in Table 10.

TABLE 10 Group Naive B1 B2 B3 B4 Challenge Amount of each antigen perN/A 150 100 50 20 N/A dose (μg/0.5 mL)

4-week-old specific pathogen-free chickens (purchased from the AnimalDrugs Inspection Branch of the Animal Health Research Institute, Councilof Agriculture, Executive Yuan, Taiwan) were used and kept in animalhouse facilities at the inspection branch. Said recombinant-antigenvaccine was administered to the chickens twice every two weeks forvaccination. Two weeks after vaccination, the chickens were challengedwith Mycoplasma synoviae MS-f1 through tracheae and footpads. Afterchallenge, blood samples were collected and serum was isolated to assessMycoplasma synoviae-specific antibodies. Weight, footpad temperature,and footpad thickness of the chickens at various time points were alsorecorded. At the end of the experiment, a dissection was performed toobserve the symptoms of air sac lesion among the chickens. Observedresults were recorded following the diagnostic criteria for organdysfunction as described in Table 5.

The results showed that the four doses of antigen vaccines used in thisexperiment could all induce antibody production in the chickens. Inaddition, the four doses that were tested in this experiment were alleffective in alleviating symptoms regarding weight and footpadtemperature (FIGS. 13 and 14). The chickens were further sacrificed anddissected. It was observed from air sac lesion scores that the fourdoses of vaccines tested were all effective in ameliorating air saclesion (see Table 12).

TABLE 11 Group Score Naive B1 B2 B3 B4 S/P ratio < 0.5 10 1 0 0 0 S/Pratio > 0.5 0 11 11 11 12 S/P positive 0 92 100 100 100 rate (%)

TABLE 12 Group Score Naive B1 B2 B3 B4 Challenge 0 12 3 3 4 6 1 1 0 3 03 4 0 2 0 3 5 3 1 5 3 0 2 2 0 1 6 Positive rate of air 0 73 70 60 50 92sac lesion (%)

1. A composition for alleviating symptoms caused by Mycoplasma synoviaeinfection, comprising: active ingredients comprising Tuf, NOX, MS53-0285or a combination thereof, wherein said Tuf comprises a sequence of SEQID NO: 01, said NOX comprises a sequence of SEQ ID NO: 02, and saidMS53-0285 comprises a sequence of SEQ ID NO: 03; and a pharmaceuticallyacceptable adjuvant.
 2. The composition of claim 1, wherein said activeingredients are at least two proteins selected from the group consistingof: Tuf, NOX, and MS53-0285.
 3. The composition of claim 1, wherein saidactive ingredients comprises Tuf, NOX, and MS53-0285.
 4. The compositionof claim 1, wherein said active ingredients have a concentration of 40μg/ML to 900 μg/mL based on the total volume of said composition.
 5. Thecomposition of claim 1, wherein said pharmaceutically acceptableadjuvant comprises a complete or incomplete Freund's adjuvant, aluminagel, surfactant, polyanion adjuvant, peptide, oil emulsion, or acombination thereof.
 6. The composition of claim 1, said compositionfurther comprising a pharmaceutically acceptable additive.
 7. Thecomposition of claim 6, wherein said pharmaceutically acceptableadditive comprises a solvent, stabilizer, diluent, preservative,antibacterial agent, antifungal agent, isotonic agent, absorptiondelaying agent, or a combination thereof.
 8. The composition of claim 1,wherein said symptoms caused by Mycoplasma synoviae infection aretracheal lesion, air sac lesion, arthritis or a combination thereof. 9.The composition of claim 8, wherein said symptoms caused by Mycoplasmasynoviae infection are at least arthritis provided that said activeingredients comprise Tuf.
 10. The composition of claim 8, wherein saidsymptoms caused by Mycoplasma synoviae infection are at least air saclesion provided that said active ingredients comprise NOX, MS53-0285, ora combination thereof.
 11. The composition of claim 8, wherein saidsymptoms caused by Mycoplasma synoviae infection are at least tracheallesion provided that said active ingredients comprise MS53-0285.
 12. Thecomposition of claim 3, wherein said symptoms caused by Mycoplasmasynoviae infection are tracheal lesion, air sac lesion and arthritis.13. An expression vector, comprising: a nucleotide sequence of SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or a combination thereof; expressionelements including a promoter and a ribosome binding site; and fusionpartner sequences.
 14. An expression vector, comprising: a nucleotidesequence translated as a protein of Tuf, NOX, MS53-0285 or a combinationthereof, wherein said Tuf has a sequence of SEQ ID NO: 01, said NOX hasa sequence of SEQ ID NO: 02, and said MS53-0285 has a sequence of SEQ IDNO: 03; expression elements including a promoter and a ribosome bindingsite; and fusion partner sequences.
 15. The expression vector of claim13, wherein said fusion partner is DsbC from E. coli, MsyB from E. coli,FklB from E. coli or a combination thereof.
 16. The expression vector ofclaim 13, wherein said expression vector has a sequence of SEQ ID NO:07, SEQ ID NO: 08, or SEQ ID NO:
 09. 17. A method for alleviatingsymptoms caused by Mycoplasma synoviae infection, said methodcomprising: administering the composition of claim 1 to a subject. 18.The expression vector of claim 14, wherein said fusion partner is DsbCfrom E. coli, MsyB from E. coli, FklB from E. coli or a combinationthereof.
 19. The expression vector of claim 14, wherein said expressionvector has a sequence of SEQ ID NO: 07, SEQ ID NO: 08, or SEQ ID NO: 09.